Professional Documents
Culture Documents
Gim201618 PDF
Gim201618 PDF
Purpose: Bosch-Boonstra-Schaaf optic atrophy syndrome two frameshifting insertions/deletions, one nonframeshifting inser-
(BBSOAS) is an autosomal-dominant disorder characterized by tion/deletion, and five whole-gene deletions). All the missense vari-
optic atrophy and intellectual disability caused by loss-of-function ants were found to impair transcriptional activity. In addition to
mutations in NR2F1. We report 20 new individuals with BBSOAS, visual and cognitive deficits, individuals with BBSOAS manifested
exploring the spectrum of clinical phenotypes and assessing potential hypotonia (75%), seizures (40%), autism spectrum disorder (35%),
genotype–phenotype correlations. oromotor dysfunction (60%), thinning of the corpus callosum (53%),
and hearing defects (20%).
Methods: Clinical features of individuals with pathogenic NR2F1
variants were evaluated by review of medical records. The functional Conclusion: BBSOAS encompasses a broad range of clinical pheno-
relevance of coding nonsynonymous NR2F1 variants was assessed types. Functional studies help determine the severity of novel NR2F1
with a luciferase assay measuring the impact on transcriptional activ- variants. Some genotype–phenotype correlations seem to exist, with
ity. The effects of two start codon variants on protein expression were missense mutations in the DNA-binding domain causing the most
evaluated by western blot analysis. severe phenotypes.
Genet Med advance online publication 17 March 2016
Results: We recruited 20 individuals with novel pathogenic NR2F1
variants (seven missense variants, five translation initiation variants, Key Words: BBSOAS; developmental delay; NR2F1; optic atrophy
The first two authors and the last two authors contributed equally to this work, and the last two authors are co–senior authors.
1
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA; 2Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital,
Houston, Texas, USA; 3Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands; 4Bartiméus, Institute for the Visually Impaired, Zeist, The
Netherlands; 5Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands; 6Department of Cognitive Neuroscience, Donders
Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands; 7GeneDx, Gaithersburg, Maryland, USA; 8Division of Genetics and
Genomic Medicine, Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA; 9Department of Ophthalmology, Baylor College of Medicine,
Houston, Texas, USA; 10Genetics, Kaiser-Permanente Fresno Medical Center, Clovis, California, USA; 11Nicklaus Children’s Hospital, Miami, Florida, USA; 12Riley Hospital for
Children, Indianapolis, Indiana, USA; 13Departments of Neurology, Medical and Molecular Genetics, and Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana,
USA; 14Genomic Medicine Institute, Cleveland Clinic, Cleveland, Ohio, USA; 15University of Minnesota Health, Minneapolis, Minnesota, USA; 16South West Thames Regional
Genetics Service, St. George’s Healthcare NHS Trust, London, UK; 17VMP Genetics, LLC, Atlanta, Georgia, USA; 18University of Georgia, Athens, Georgia, USA; 19Center for Rare
Childhood Disorders, Translational Genomics Research Institute, Phoenix, Arizona, USA; 20Department of Pediatrics, Columbia University Medical Center, New York, New York,
USA; 21Nyack Hospital, Nyack, New York, USA; 22Department of Medicine, Columbia University Medical Center, New York, New York, USA; 23Division of Medical Genetics,
Department of Pediatrics, Ochsner Clinic Foundation, New Orleans, Louisiana; 24Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical
Center, Dallas, Texas, USA; 25Division of Pediatric Neurology, Akron Children’s Hospital, Akron, Ohio, USA; 26McMaster University Medical Center, Hamilton, Ontario, Canada;
27
Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, Washington, USA; 28Texas Children’s Hospital, Houston, Texas, USA. Correspondence:
Bert B.A. de Vries and Christian P. Schaaf (Bert.deVries@radboudumc.nl; Schaaf@bcm.edu)
Submitted 15 October 2015; accepted 19 January 2016; advance online publication 17 March 2016. doi:10.1038/gim.2016.18
a DNA-binding domain (DBD), formed by two zinc-finger fibroblasts were frozen at that time. Upon receipt of signed con-
domains, and a putative ligand-binding domain.3 The physio- sent from the individual’s parents, fibroblasts were transferred
logical functions of NR2F1 have been studied extensively thanks to Dr Schaaf ’s laboratory for further testing.
to an Nr2f1 knockout mouse model (the human NR2F1 and
murine Nr2f1 proteins share 99% amino acid identity). Previous Tissue culture
studies showed that Nr2f1 is important for cortical patterning,4–7 HEK293T cells were purchased from ATCC (Manassas, VA). Control
thalamocortical axon guidance,8 neurogenesis,8 arborization,9 human fibroblast cells and human lymphoblast cells were from the
and eye and optic nerve development.10,11 By contrast, studies of cell culture core at the Baylor College of Medicine Intellectual and
human disorders resulting from loss-of-function NR2F1 muta- Developmental Disabilities Research Center. HEK293T cells and
tions have been limited.1,12,13 Moreover, these studies may not be human fibroblast cells were grown in Dulbecco’s modified Eagle
sufficient to describe the full phenotypic spectrum of BBSOAS medium (Corning Cellgro, Manassas, VA). Human lymphoblast
because of the small number of cases identified so far. Here we cells were grown in RPMI 1640 medium (Invitrogen, Waltham,
report 20 new individuals with pathogenic NR2F1 variants and MA). Both media were supplemented with 10% fetal bovine serum
summarize their clinical features. and 1% penicillin-streptomycin (Invitrogen, Waltham, MA). All
cell cultures were maintained at 37 °C in a humidified incubator
MATERIALS AND METHODS supplemented with 5% carbon dioxide.
Human subjects
Individuals with probable pathogenic NR2F1 variants were Dual-luciferase reporter assay
enrolled on the basis of their genotypes. From clinical whole- Three expression vectors were used in the assay. The
exome sequencing data, we selected coding nonsynonymous pXP2-NGFI-A vector expresses firefly luciferase under control
variants that were not present in the ESP5400 Exome Variant of the NR2F1-activated promoter, NGFI-A (−168/+33), kindly
Server and that were predicted to be damaging/probably damag- provided by Ming-Jer Tsai.10 The pRL-TK vector expresses
ing by at least one algorithm (PolyPhen-2/Mutation Taster). All Renilla luciferase as an internal control reporter and was pur-
these were confirmed by Sanger sequencing. Whole-gene dele- chased from Promega. Both the pXP2-NGFI-A and pRL-TK
tion mutations were identified based on clinical chromosome vectors were cotransfected with expression plasmid pcDNA5 of
microarray analysis. Three individuals (individuals 1, 3, and either wild-type or mutant mouse Nr2f1 into HEK293T cells.
11) were enrolled through Baylor Miraca Genetics Laboratories All point mutations were generated by a QuikChange site-
and eight (individuals 2, 5, 7, 8, 10, 12, 14, and 15) through the directed mutagenesis kit (Agilent, Santa Clara, CA). HEK293T
GeneDx laboratory. Upon identification of a probable patho- cells were seeded in a 24-well plate 24 h before transfection.
genic variant in NR2F1, genetics counselors from the respective Cells were cotransfected with 5 ng of each expression vector
laboratories contacted the referring providers to inform them (total of three) per well with Lipofectamine 2000 following the
of research interest by Dr Schaaf ’s laboratory. Four other indi- manufacturer’s instructions. Cells were harvested 48 h after
viduals (individuals 9, 16, 17, and 18) were enrolled through the transfection, and the luciferase activity was measured following
Radboud University Medical Center, Nijmegen, the Netherlands. the manufacturer’s instructions (Promega, Fitchburg, WI). The
Individual 9 has been reported before,14 and re-analysis of the luciferase activity was quantified by normalizing firefly lucifer-
whole-exome sequencing data showed a low covered de novo ase reads to Renilla luciferase reads. The luciferase activity of
NR2F1 variant, which was validated by Sanger sequencing. The wild-type Nr2f1 and the empty vector was set as 100 and 0%,
referring providers of individuals 16, 17, and 18 contacted Dr de respectively. Results are mean values ± SEM from three inde-
Vries. For the rest of the individuals (individuals 4, 6, 13, 19, and pendent experiments performed in triplicate.
20), the referring provider or family contacted Dr Schaaf directly
because of a new diagnosis of BBSOAS made by genetic testing. Western blot analysis
All families reported herein agreed to share clinical informa- Human patient or control fibroblast cell lines were harvested
tion and were enrolled under a research protocol approved by (1.3 × 106 cells) and lysed with modified radioimmunoprecipi-
the Institutional Review Board of Baylor College of Medicine. tation assay buffer (25 mmol/l Tris-HCl (pH 7.8), 150 mmol/l
Medical records were reviewed, and the referring providers NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium
were asked to complete a clinical questionnaire. All families who dodecyl sulfate, and complete protease inhibitor cocktail
shared photographs signed consent for publication. (Roche, Indianapolis, IN)). Protein lysate was collected after
boiling for 5 min, followed by centrifugation at 13,000 rpm for
Generation of patient fibroblast cell lines 10 min. Proteins were quantified using a Pierce BCA Protein
Individuals 13 and 14, who had a probable pathogenic variant Assay Kit (Thermo Scientific, Waltham, MA). Protein (15 μg)
affecting the start codon of NR2F1, had a skin biopsy performed was resolved by electrophoresis on a NuPAGE 4–12% bis-Tris
to establish a fibroblast culture. Individual 13 consented to par- Gel (Life Technologies, Waltham, MA) and transferred to
ticipate in a research protocol approved by the Institutional nitrocellulose membranes. Blots were blocked in 5% skim milk
Review Board of Baylor College of Medicine. Individual 14 had and incubated with anti-COUP-TFI antibody (1:1,000 dilu-
a skin biopsy performed in 2004 for diagnostic purposes, and tion; catalog no. 6364S; Cell Signaling Technologies, Beverly,
a
M1? indel C128R
M1? (c.2T>C) ×2 R135S
M1? (c.2T>G) ×2 Phe110del C138Y
R142L
H97fs
C146R G368D
G35fs A155T
N * * DBD * LBD C
b 150
Relative luciferase activity
100
*
50 **
** ** ** ** ** ** **
** ** ** ** ** **
0
−50
wt
110 del
C128R
C135S
C138Y
R142L
R146R
R155T
G368D
Figure 1 Pathogenic NR2F1 variants decrease transcriptional activity. (a) Distribution of NR2F1 point mutations in patients with Bosch-Boonstra-Schaaf
optic atrophy syndrome. Most of the mutations (except p.Ala155Thr) fall into the DNA-binding domain or ligand-binding domain. The number of individuals
who share the same translation initiation variant is indicated. (b) In vitro dual-luciferase reporter assay for NR2F1 point mutations. HEK293T cells cotransfected
with dual-luciferase vectors (pXP2-NGFI-A and pRL-TK vectors) and either wild-type or mutant Nr2f1 expression vectors were analyzed. The luciferase activity
of wild-type Nr2f1 and the empty vector are set as 100 and 0%, respectively. Data are means ± SEM (n = 3). *P < 0.05, ****P < 0.0001.
p.Ala155Thr), which is located outside of, but adjacent to, the a Control p.M1?
DBD. This variant was predicted to be pathogenic by Mutation
4
l1
l1
Taster but not PolyPhen-2. The luciferase assay showed that this
l1
l2
ua
ua
tro
tro
id
id
v
v
mutation significantly decreased reporter activity, but only by
on
on
di
di
In
In
C
C
30%, which is less dramatic than the decrease in activity result- NR2F1
ing from missense substitutions in the DBD itself. Finally, we
confirmed that one nonframeshifting indel within the DBD
GAPDH
(individual 8, p.Phe110del) also reduced reporter activity.
Collectively, the enrichment of pathogenic mutations in the 1.5
DBD and ligand-binding domain indicates the importance of
these highly conserved functional domains in NR2F1.
1147
Original Research Article CHEN et al | The expanding clinical phenotype of BBSOAS
5.0 Mb
1.2 Mb
0.9 Mb
0.9 Mb
0.2 Mb
Figure 3 Overview of the microdeletions encompassing the NR2F1 gene from patients with Bosch-Boonstra-Schaaf optic atrophy syndrome.
The sizes of the microdeletions range from 0.2 to 5.0 Mb. Individuals 17 and 18 are a father and son who share the same microdeletion containing NR2F1.
Figure 4 Facial appearance of patients with Bosch-Boonstra-Schaaf optic atrophy syndrome. While some individuals manifest mild dysmorphic facial
features, they do not appear to be consistent between individuals.
diagnosis of autism spectrum disorder. A history of seizures impairment, and 5% (1/20) with spasticity. While some indi-
was present in 40% (8/20); two of these eight individuals had viduals manifest mild dysmorphic facial features, they do
a history of infantile spasms. In addition, one individual had not seem to be consistent between individuals (Figure 4). In
had a single febrile seizure. Other clinical features were pres- summary, we showed that NR2F1 haploinsufficiency leads
ent at lower prevalences, including 25% (5/20) with atten- not only to cognitive deficits and visual impairment but also
tion deficit hyperactivity disorder, 20% (4/20) with hearing to a much broader range of phenotypic features, including
As the number of individuals identified with BBSOAS con- 4. Faedo A, Tomassy GS, Ruan Y, et al. COUP-TFI coordinates cortical patterning,
neurogenesis, and laminar fate and modulates MAPK/ERK, AKT, and beta-
tinues to grow, families are beginning to connect via social catenin signaling. Cereb Cortex 2008;18:2117–2131.
media (https://www.facebook.com/pages/NR2F1-Collabora 5. Armentano M, Chou SJ, Tomassy GS, Leingärtner A, O’Leary DD, Studer M.
tive/434582583373135). To gain further insight into the phe- COUP-TFI regulates the balance of cortical patterning between frontal/motor
and sensory areas. Nat Neurosci 2007;10:1277–1286.
notypic spectrum of BBSOAS, a Web-based database has been 6. Zhou C, Tsai SY, Tsai MJ. COUP-TFI: an intrinsic factor for early regionalization of
developed so that the phenotypes of individuals with NR2F1 the neocortex. Genes Dev 2001;15:2054–2059.
aberrations can be updated (http://www.nr2f1gene.com). 7. Alfano C, Magrinelli E, Harb K, Hevner RF, Studer M. Postmitotic control of sensory
area specification during neocortical development. Nat Commun 2014;5:5632.
8. Zhou C, Qiu Y, Pereira FA, Crair MC, Tsai SY, Tsai MJ. The nuclear orphan
SUPPLEMENTARY DATA receptor COUP-TFI is required for differentiation of subplate neurons and
Supplementary information is linked to the online version of the guidance of thalamocortical axons. Neuron 1999;24:847–859.
paper at http://www.nature.com/gim 9. Qiu Y, Pereira FA, DeMayo FJ, Lydon JP, Tsai SY, Tsai MJ. Null mutation of
mCOUP-TFI results in defects in morphogenesis of the glossopharyngeal
ganglion, axonal projection, and arborization. Genes Dev 1997;11:1925–1937.
ACKNOWLEDGMENTS 10. Tang K, Xie X, Park JI, Jamrich M, Tsai S, Tsai MJ. COUP-TFs regulate eye
C.P.S. is generously supported by the Joan and Stanford Alexander development by controlling factors essential for optic vesicle morphogenesis.
Development 2010;137:725–734.
family. C.P.S. has received a Clinical Scientist Development Award 11. Satoh S, Tang K, Iida A, et al. The spatial patterning of mouse cone opsin
from the Doris Duke Charitable Foundation. R.A.L. is a senior expression is regulated by bone morphogenetic protein signaling through
scientific investigator for Research to Prevent Blindness, whose downstream effector COUP-TF nuclear receptors. J Neurosci 2009;29:
12401–12411.
unrestricted funds to his department support part of these stud-
12. Brown KK, Alkuraya FS, Matos M, Robertson RL, Kimonis VE, Morton CC.
ies. This research is supported by the Intellectual and Develop- NR2F1 deletion in a patient with a de novo paracentric inversion, inv(5)
mental Disabilities Research Center (1U54 HD083092), Stichting (q15q33.2), and syndromic deafness. Am J Med Genet A 2009;149A:
ODAS (to F.N.B. and F.P.M.C.), Vereniging Bartiméus-Sonneheerdt 931–938.
13. Al-Kateb H, Shimony JS, Vineyard M, Manwaring L, Kulkarni S, Shinawi M.
(5781251 to F.N.B. and F.P.M.C.), Oogfonds (to F.P.M.C., F.N.B., NR2F1 haploinsufficiency is associated with optic atrophy, dysmorphism and
and B.B.A.deV.), and LSBS (to F.P.M.C., F.N.B., and B.B.A.deV.). The global developmental delay. Am J Med Genet A 2013;161A:377–381.
authors are grateful to the individuals and their families for their 14. Bosch DG, Boonstra FN, de Leeuw N, et al. Novel genetic causes for cerebral
visual impairment. Eur J Hum Genet; e-pub ahead of print 9 September 2015.
support and for participating in our research study. They thank 15. Michaud JL, Lachance M, Hamdan FF, et al. The genetic landscape of infantile
Ming-Jer Tsai and Mafei Xu for providing technical assistance and spasms. Hum Mol Genet 2014;23:4846–4858.
Huda Y. Zoghbi for valuable input, guidance, and discussion. 16. Hino-Fukuyo N, Kikuchi A, Arai-Ichinoi N, et al. Genomic analysis identifies
candidate pathogenic variants in 9 of 18 patients with unexplained West
syndrome. Hum Genet 2015;134:649–658.
DISCLOSURE 17. Dimassi S, Labalme A, Ville D, et al. Whole-exome sequencing improves the
W.K.C. is a consultant for BioReference Laboratories. The other diagnosis yield in sporadic infantile spasm syndrome. Clin Genet 2016;89:198–
authors declare no conflict of interest. 204.
18. Panicker IS, Browning GF, Markham PF. The effect of an alternate start codon on
heterologous expression of a phoA fusion protein in Mycoplasma gallisepticum.
References PLoS One 2015;10:e0127911.
1. Bosch DG, Boonstra FN, Gonzaga-Jauregui C, et al.; Baylor-Hopkins Center for 19. Cooney AJ, Tsai SY, O’Malley BW, Tsai MJ. Chicken ovalbumin upstream
Mendelian Genomics. NR2F1 mutations cause optic atrophy with intellectual promoter transcription factor (COUP-TF) dimers bind to different GGTCA
disability. Am J Hum Genet 2014;94:303–309. response elements, allowing COUP-TF to repress hormonal induction of
2. Sagami I, Tsai SY, Wang H, Tsai MJ, O’Malley BW. Identification of two factors the vitamin D3, thyroid hormone, and retinoic acid receptors. Mol Cell Biol
required for transcription of the ovalbumin gene. Mol Cell Biol 1986;6: 1992;12:4153–4163.
4259–4267. 20. Armentano M, Filosa A, Andolfi G, Studer M. COUP-TFI is required for the
3. Lin FJ, Qin J, Tang K, Tsai SY, Tsai MJ. Coup d’Etat: an orphan takes control. formation of commissural projections in the forebrain by regulating axonal
Endocr Rev 2011;32:404–421. growth. Development 2006;133:4151–4162.